Emmanuelle Wilhelm

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

BackgroundHIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown.ResultsUsing electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat.ConclusionOur data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

HMGA1 directly interacts with TAR to modulate basal and Tat-dependent HIV transcription

The transactivating response element (TAR) of human immunodeficiency virus 1 (HIV-1) is essential for promoter transactivation by the viral transactivator of transcription (Tat). The Tat-TAR interaction thereby recruits active positive transcription elongation factor b (P-TEFb) from its inactive, 7SK/HEXIM1-bound form, leading to efficient viral transcription. Here, we show that the 7SK RNA-associating chromatin regulator HMGA1 can specifically bind to the HIV-1 TAR element and that 7SK RNA can thereby compete with TAR. The HMGA1-binding interface of TAR is located within the binding site for Tat and other cellular activators, and we further provide evidence for competition between HMGA1 and Tat for TAR-binding. HMGA1 negatively influences the expression of a HIV-1 promoter-driven reporter in a TAR-dependent manner, both in the presence and in the absence of Tat. The overexpression of the HMGA1-binding substructure of 7SK RNA results in a TAR-dependent gain of HIV-1 promoter activity similar to the effect of the shRNA-mediated knockdown of HMGA1. Our results support a model in which the HMGA1/TAR interaction prevents the binding of transcription-activating cellular co-factors and Tat, subsequently leading to reduced HIV-1 transcription.

TAF6δ Controls Apoptosis and Gene Expression in the Absence of p53

Background Life and death decisions of metazoan cells hinge on the balance between the expression of pro- versus anti-apoptotic gene products. The general RNA polymerase II transcription factor, TFIID, plays a central role in the regulation of gene expression through its core promoter recognition and co-activator functions. The core TFIID subunit TAF6 acts in vitro as an essential co-activator of transcription for the p53 tumor suppressor protein. We previously identified a splice variant of TAF6, termed TAF6δ that can be induced during apoptosis. Methodology/Principal Findings To elucidate the impact of TAF6δ on cell death and gene expression, we have employed modified antisense oligonucleotides to enforce expression of endogenous TAF6δ. The induction of endogenous TAF6δ triggered apoptosis in tumor cell lines, including cells devoid of p53. Microarray experiments revealed that TAF6δ activates gene expression independently of cellular p53 status. Conclusions Our data define TAF6δ as a pivotal node in a signaling pathway that controls gene expression programs and apoptosis in the absence of p53.

Determining the impact of alternative splicing events on transcriptome dynamics

BackgroundThe complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events.ResultsWhen applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis.ConclusionThe strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression.

TAF6δ orchestrates an apoptotic transcriptome profile and interacts functionally with p53

BackgroundTFIID is a multiprotein complex that plays a pivotal role in the regulation of RNA polymerase II (Pol II) transcription owing to its core promoter recognition and co-activator functions. TAF6 is a core TFIID subunit whose splice variants include the major TAF6α isoform that is ubiquitously expressed, and the inducible TAF6δ. In contrast to TAF6α, TAF6δ is a pro-apoptotic isoform with a 10 amino acid deletion in its histone fold domain that abolishes its interaction with TAF9. TAF6δ expression can dictate life versus death decisions of human cells.ResultsHere we define the impact of endogenous TAF6δ expression on the global transcriptome landscape. TAF6δ was found to orchestrate a transcription profile that included statistically significant enrichment of genes of apoptotic function. Interestingly, gene expression patterns controlled by TAF6δ share similarities with, but are not equivalent to, those reported to change following TAF9 and/or TAF9b depletion. Finally, because TAF6δ regulates certain p53 target genes, we tested and demonstrated a physical and functional interaction between TAF6δ and p53.ConclusionTogether our data define a TAF6δ-driven apoptotic gene expression program and show crosstalk between the p53 and TAF6δ pathways.

Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication

Abstract We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2β, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2β, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents.

Alternative Splicing of TAF6: Downstream Transcriptome Impacts and Upstream RNA Splice Control Elements

The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ.

Probing Endogenous RNA Polymerase II Pre-initiation Complexes by Electrophoretic Mobility Shift Assay

RNA polymerase II (Pol II) plays a crucial role in eukaryotic biology since it is necessary for the expression of all protein-coding genes as well as most microRNAs and several small nuclear RNAs. Pol II is specifically recruited to core promoter DNA via its association with general transcription factors (GTFs) that possess DNA binding activity such as TFIID, TFIIA, and TFIIB. The large multi-protein assemblies of Pol II together with the GTFs required for productive transcription are termed pre-initiation complexes (PICs). To date, studies of the interaction of PICs with promoter DNA have relied on the use of purified or recombinant GTFs. Recent findings have demonstrated an astonishing diversity in the function of core promoters as well as in the protein composition of PICs. The currently known subset of GTFs alone cannot account for observed PIC and core promoter diversity. In order to identify the full complement of factors that impart PIC specificity, techniques to analyze the DNA binding of endogenous PIC are essential. Analysis of endogenous PIC formation has remained out of reach due to technical hurdles presumably including the large size of endogenous PIC, their highly dynamic association with core promoters, and the complex topology of DNA bound to PIC. We have optimized electrophoretic mobility shift assays (EMSAs) to achieve the detection of endogenous Pol II PIC from nuclear extracts of human cells. Here, we provide a robust and sensitive EMSA method for the analysis of endogenous Pol II PICs.

Inhibition of NF-κB-dependent HIV-1 replication by the marine natural product bengamide A

ABSTRACT HIV‐1 inhibitors that act by mechanisms distinct from existing antiretrovirals can provide novel insights into viral replication and potentially inform development of new therapeutics. Using a multi‐cycle HIV‐1 replication assay, we screened 252 pure compounds derived from marine invertebrates and microorganisms and identified 6 (actinomycin Z2, bastadin 6, bengamide A, haliclonacyclamine A + B, keramamine C, neopetrosiamide B) that inhibited HIV‐1 with 50% effective concentrations (EC50s) of 3.8 &mgr;M or less. The most potent inhibitor, bengamide A, blocked HIV‐1 in a T cell line with an EC50 of 0.015 &mgr;M and in peripheral blood mononuclear cells with an EC50 of 0.032 &mgr;M. Bengamide A was previously described to inhibit NF‐&kgr;B signaling. Consistent with this mechanism, bengamide A suppressed reporter expression from an NF‐&kgr;B‐driven minimal promoter and an HIV‐1 long terminal repeat (LTR) with conserved NF‐&kgr;B response elements, but lacked activity against an LTR construct with mutation of these elements. In single‐cycle HIV‐1 infection assays, bengamide A also suppressed viral protein expression when viruses encoded an intact LTR but exhibited minimal activity against those with mutated NF‐&kgr;B elements. Finally, bengamide A did not inhibit viral DNA accumulation, indicating that it likely acts downstream of this step in HIV‐1 replication. Our study identifies multiple new antiviral compounds including an unusually potent inhibitor of HIV‐1 gene expression. Graphical abstract Figure. No caption available. Highlights6 novel HIV‐1 inhibitors were identified from a pure marine natural products library.The most active compound, bengamide A, inhibits HIV‐1 in cell lines and primary cells with sub‐micromolar efficacy.The HIV‐1 LTR NF‐&kgr;B response elements are required for Bengamide A‐mediated inhibition of LTR‐dependent gene expression.An HIV‐1 strain with mutated LTR NF‐kB sites exhibits in vitro resistance to bengamide A.

Selective recognition of viral promoters by host cell transcription complexes: challenges and opportunities to control latency

The rate of transcription driven by the HIV promoter defines both the entry into and reactivation from viral latency. The HIV core promoter plays a pivotal role in HIV latency by recruiting host cell RNA polymerase II pre-initiation complexes essential for viral transcription. Pioneering studies on the HIV core promoter revealed that the architecture of the HIV core promoter is specifically required for the amplification of transcription in response to the viral trans-activator Tat, and provided the proof-of-concept that the HIV core promoter represents a tractable drug target. The recent discovery of host cell transcription complexes that selectively recognize the HIV core promoter provides new impetus to investigate their components as novel targets to therapeutically extinguish or eradicate latent HIV.